Method of regulating a skin condition

ABSTRACT

A method of regulating a skin conditions that includes applying a safe and effective amount of achachairu to a target portion of skin in need of treatment or where treatment is desired. The achachairu can be in the form of a bioactive ingredient that exhibits anti-inflammatory, anti-oxidant, and/or anti-aging properties, and which is free or substantially free of benzophenones and/or protein. The achachairu ingredient may be a serum fraction and/or an extract, and may be incorporated into a skin care composition formulated for topical use on skin.

FIELD

The present invention relates generally to regulating a skin conditionwith achachairu. More specifically, the present invention relates tousing achachairu serum fractions and/or extracts to regulate a skincondition, for example, by incorporating the fraction or extract into askin care composition.

BACKGROUND

Human skin is constantly subjected to a variety of insults fromextrinsic and intrinsic sources. Extrinsic sources include ultravioletradiation, environmental pollution, wind, heat, infrared radiation, lowhumidity, harsh surfactants, abrasives, etc, whereas intrinsic sourcesinclude chronological aging and other biochemical changes from withinthe body. Whether extrinsic or intrinsic, these factors can result invisible signs of damage (e.g., fine lines, wrinkling, hyperpigmentation,sallowness, sagging, dark under-eye circles, puffy eyes, enlarged pores,diminished rate of skin cell turnover, flaking, scaling, dryness,roughness). Currently, there are a number of skin care productsavailable to consumers, the majority of which are directed to delaying,minimizing or even reversing the changes associated with aging skinand/or environmental damage to skin. However, there is a continuing needfor products and methods that seek to remedy undesirable skinconditions.

One potential source of bioactive ingredients for regulating conditionsin mammalian keratinous tissue is achachairu. Achachairu (Garciniahumilis) is a plant belonging to the Clusiaceae (or Guttiferae) familyand is widely distributed in the region of Santa Cruz, Bolivia. Theachachairu plant (e.g., fruit) is used in Bolivian folk medicine for itshealing, digestive, and laxative properties. In Brazil, achachairu ispopularly known as “achacha” and is used in folk medicine to treatrheumatism, inflammation, pain and gastric disorders (Alves T M A, SilvaA F, Brandao M, Grandi T S M, Smania E F et al. (2000) BiologicalScreening of Brazilian Medicinal Plants. Mem Inst Oswaldo Cruz 95:367-373 and Barbosa W, Chagas E A, Martins L, Pio R, Tucci M L et al.(2008) “Germinação de sementes e desenvolvimento inicial de plântulas deachachairu. Rev Bras Frutic.” 30: 263-266).

Some studies suggest that achachar, like many plants, contains unwantedcomponents of concern, such as benzophenones (e.g., guttiferone A) andproteins. See, e.g., Acuña U M, et al., (2009) Polyisoprenylatedbenzophenones from Clusiaceae: potential drugs and lead compounds. CurrTop Med Chem 9: 1560-1580). Benzophenones are known to exhibit variousbiological activities such as cytotoxic, genotoxic antimicrobial,antiviral and antioxidant activity. See, e.g., Terrazas P M, et al.(2013) “Benzophenone guttiferone A from Garcinia achachairu Rusby(Clusiaceae) Presents Genotoxic Effects in Different Cells of Mice;”PLoS ONE 8(11): e76485). And proteins are known to cause allergicreactions when contacted with skin. See, e.g., V. Janssens, et al.,(2015) “Protein contact dermatitis: myth or reality?” British Journal ofDermatology; 132: 1-6). Thus, in some instances it may be desirable toremove these unwanted components or reduce their concentrationssubstantially when providing an achachairu ingredient for incorporationinto a skin care composition.

SUMMARY OF THE INVENTION

Disclosed herein is a method of regulating a skin condition, whichcomprises identifying a target portion of skin in need of treatment orwhere treatment is desired; and applying an effective amount ofachachairu serum fraction to the target portion of skin during atreatment period, wherein the treatment period is sufficient for theachachairu serum fraction to provide a skin care benefit.

Also disclosed is a method of providing skin care benefit, whichcomprises identifying a target portion of keratinous tissue in need oftreatment; and applying an effective amount of an achachairu extract tothe target portion of skin during a treatment period, wherein thetreatment period is sufficient for the achachairu extract to provide askin care benefit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a cross-sectional view of an achachairu fruit.

FIG. 2 is a schematic of a process for preparing bioactive serumfractions and bioactive fraction extracts derived from achachairu.

FIGS. 3A, 3B, 4A, 4B are graphs illustrating absorbance spectra ofachachairu serum fractions and extracts.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that achachairu (Garcinia humilis),especially bioactive extracts and serum fractions obtained fromparticular portions of the achachairu plant, possess properties that maybe beneficial for regulating certain skin conditions. In particular,bioactive achachairu fruit extracts and serum fractions exhibitsurprising properties related to anti-oxidation, inhibiting melaninproduction, and inhibiting lipogenesis, which can be exploited toprovide a wide variety of different skin health benefits. Thus, it isnow possible to regulate a particular skin condition by applying aneffective amount of achachairu to a target portion of skin wheretreatment is needed or desired.

It may also be desirable to provide achachairu serum fractions and/orextracts that are free or substantially free of benzonphenones (e.g.,guttiferone A) and/or proteins. As used herein, “substantially free ofproteins” means less than 0.15% total protein content determined byhydrolyzed and un-hydrolyzed amino acid analysis conducted on an HitachiL-8900 amino acid analyzer or equivalent, and “substantially free ofbenzophenones” means less than 0.1% total benzophenones, includingguttiferone A, as determined by the method described in Dal Molin M M,et al., (2012) “Phytochemical analysis and antinociceptive properties ofGarcinia achachairu Rusby (Clusiaceae) seeds.” Arch Pharm Res 35:623-631.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” are intended to include the plural forms as well,unless the context clearly indicates otherwise. All percentagesdisclosed herein are by weight of the total composition, unlessspecifically stated otherwise. All ratios are weight ratios, unlessspecifically stated otherwise. The number of significant digits conveysneither a limitation on the indicated amounts nor on the accuracy of themeasurements. All numerical amounts are understood to be modified by theword “about” unless otherwise specifically indicated. All measurementsare understood to be made at 25° C. and at ambient conditions, where“ambient conditions” means conditions under about one atmosphere ofpressure and at about 50% relative humidity. All such weights as theypertain to listed ingredients are based on the active level and do notinclude carriers or by-products that may be included in commerciallyavailable materials, unless otherwise specified. All numeric ranges areinclusive of narrower ranges and combinable; delineated upper and lowerrange limits are interchangeable to create further ranges not explicitlydelineated.

The compositions of the present invention can comprise, consistessentially of, or consist of, the essential components as well asoptional ingredients described herein. As used herein, “consistingessentially of” means that the composition or component may includeadditional ingredients, but only if the additional ingredients do notmaterially alter the basic and novel characteristics of the claimedcompositions or methods.

Definitions

“About,” as used herein, modifies a particular value, by referring to arange equal to the particular value, plus or minus twenty percent(+/−20%) or less (e.g., less than 15%, 10%, or even less than 5%).

“Apply” or “application”, as used in reference to a composition, meansto apply or spread the compositions of the present invention onto ahuman skin surface such as the epidermis.

“Derivative” means a molecule similar to that of another one, butdiffering from it with respect to a certain functional moiety (e.g.,esters, ethers, amides, amines, carboxylic acids, hydroxyls, acetyls,thiols, halogens, thiols, and/or salt derivatives of the relevantmolecule).

“Dermatologically acceptable” means that the compositions or componentsdescribed are suitable for use in contact with human keratinous tissuewithout undue toxicity, incompatibility, instability, allergic response,and the like.

“Effective amount” means an amount of a compound or compositionsufficient to significantly induce a positive benefit to skin, such as ahealth, appearance, and/or feel benefit, including, independently or incombination, the benefits disclosed herein, but low enough to avoidserious side effects (i.e., to provide a reasonable benefit to riskratio, within the scope of sound judgment of the skilled artisan). An“effective amount of achachairu” is an amount of achachairu sufficientto regulate a desired skin condition of mammalian keratinous tissue whentopically applied thereto in a skin care composition.

“Exogenous solvent” means any solvent placed in contact with the plantmaterial for the purpose of separating compounds from the plantmaterial. It may be a solvent originally present in plant material butadded in higher amounts (e.g., in case of water extracts), or a solventnot inherently present in plant material. Plant material herein caninclude any solid or liquid portion of the achachairu plant.

“Extract,” when referring to achachairu, means a material (e.g., one ormore phytochemicals) obtained from a portion of achachairu plantmaterial (e.g., peel, fruit, seed, stem, bark, leaves, roots, and/or acombination of these), which may be fresh, dried, or partially dried, bycontacting the plant portion(s) with an exogenous solvent and separatingthe desired material from the solvent using a conventional extractionprocess. Conventional extraction processes for obtaining plant extractsare well known in the art.

“Regulating a skin condition,” as used herein, means improving thehealth, appearance, and/or feel of skin.

“Salts” refer to ionic forms of a given compound combined withcounterions deemed acceptable for a given application (e.g., food,topical, pharmaceutical). Examples include but are not limited tosodium, potassium, calcium, ammonium, manganese, copper, and/ormagnesium salts of a given compound.

“Skin care” means regulating and/or improving a skin condition. Somenon-limiting examples include improving skin appearance and/or feel byproviding a smoother, more even appearance and/or feel; increasing thethickness of one or more layers of the skin; improving the elasticity orresiliency of the skin; improving the firmness of the skin; and reducingthe oily, shiny, and/or dull appearance of skin, improving the hydrationstatus or moisturization of the skin, improving the appearance of finelines and/or wrinkles, improving skin exfoliation or desquamation,plumping the skin, improving skin barrier properties, improving skintone, reducing the appearance of redness or skin blotches, and/orimproving the brightness, radiancy, or translucency of skin.

“Skin care active” means an active that, when applied to skin, providesan acute and/or chronic benefit to skin or a type of cell commonly foundtherein. Skin care actives may regulate and/or improve skin or itsassociated cells (e.g., improve skin elasticity, hydration, skin barrierfunction, and/or cell metabolism).

“Skin care composition” means a composition that includes a skin careactive and regulates and/or improves a skin condition.

“Topical” refers to a composition that is intended to be applied to abodily surface such as skin or hair.

Skin Care Composition

The skin care compositions suitable for use in the methods hereininclude a safe and effective amount of achachairu (Garcinia humilis).The achachairu may be in form of a serum fraction, an extract, a powder,a juice, or a combination thereof. The achachairu may be obtained fromany part of the Garcinia humilis plant, as desired, but it may beparticularly desirable to use the achachairu fruit or a portion thereof(e.g., peel, pulp, and/or seed). Some testing suggests that theachachairu fruit, especially the peel, may contain higher concentrationsof biologically active compounds (e.g., carbohydrates, citric acid,flavonoids, xanthones and tannins) than other parts of the achachairuplant for regulating particular conditions of mammalian keratinoustissue. The achachairu extract and/or serum fraction may be obtainedaccording to the methods described in more detail below and combinedwith a dermatologically acceptable carrier, along with any optionalingredients, using conventional methods of making skin carecompositions. The personal care composition may then be packaged forcommercial distribution.

FIG. 1 illustrates the distinct layers and parts of the achachairufruit, as shown by a section passing through a long axis of the fruit.Whole fruit, a particular part of fruit, or any combination of parts offruit may be used to provide a serum fraction or extract herein.

However, this is not meant to exclude further distinctions of fruitparts than described here, or leaves that may be retained duringharvesting. As shown in FIG. 1, the outermost layer of the achachairufruit is the “Peel”, which is comparatively thick and tough. The nextinward layer is the “Flesh”, which is comparatively soft and yielding.The flesh surrounds a comparatively large “Seed”. Especially large fruitmay have more than one seed.

The amount of achachairu that is “effective” can differ from oneparticular source (e.g., manufacturer) to another, and can be determinedby the skilled artisan based upon a particular product's level ofactivity (e.g., level of active components present). The concentrationof active components in the particular achachairu product to be usedwill depend on factors such as the final dilution volume of the product,the particular method employed, the natural range of variation amongindividual plants, and other common factors known to those skilled inthe art. In some instances, an effective amount of achachairu may rangefrom 0.01% to 15% (e.g., from 0.1% to 10%, 0.2% to 7%, 0.5% to 5%, oreven 1% to 3%).

The skin care compositions herein are intended for topical applicationto skin, and are formulated accordingly (e.g., have suitable viscosity,color, fragrance, and feel properties). The present skin carecompositions may be provided in a variety of forms, including, but notlimited to, emulsions, lotions, milks, liquids, solids, creams, gels,mousses, ointments, pastes, serums, sticks, sprays, tonics, aerosols,foams, and/or pencils.

FIG. 2 illustrates an exemplary method for providing bioactiveachachairu ingredients. It is to be appreciated that portions of theprocess referring to “fruit” can include any portion of the achachairuplant, as desired. The individual steps illustrated in FIG. 2 aredescribed in more detail below.

“Cleaning” involves the removal of debris from the harvested achachairuplant material, prior to further processing, in a way that avoids injuryto the fruit or removal of juice or other desirable components. Forexample, cleaning can be performed by low-pressure rinsing with potablewater, under conditions where residual water wash would not noticeablycontain plant pigments.

“Selection of material” involves separating various parts of theachachairu plant material to further process a particular part alone, orin combination with other parts. This includes, but is not limited to:selecting whole fruit, peel, flesh, seed and/or leaves for furtherprocessing.

“Maceration” is an optional step for rendering the plant material and/orselected parts of the plant material into smaller particles andotherwise disrupting the integrity of the fruit to ease the followingexpelling of liquid juice. If the selected plant material is especiallyyielding (such as the flesh of the fruit) or otherwise deemed suitablefor conditions and equipment of the pressing step, maceration may beomitted. Examples of suitable maceration implements include, but are notlimited to, devices such as a crusher, a grinder, or a mill (e.g., knifemill or hammer mill). To prevent temperature-induced degradation ofplant material, maceration can include temperature monitoring andselection of maceration parameters which minimize the risk oftemperature increase during this step.

“Pressing” involves the application of mechanical force to the selected,and optionally macerated, achachairu plant material to separate at leastsome of the liquid component from the rest of the plant material. Theapplied mechanical force may be provided by any suitable means known inthe art (e.g., ambient gravity, centrifugal force from a rotaryexpeller, pressure from the piston of a hydraulic press, or rollers or ascrew of appropriate type of press).

“Mechanical separation” involves the removal of relatively largeachachairu particles (e.g., pulp or agglomerated/aggregated cloudparticles) from the liquid component of the achachairu plant material.

“Juice” refers to the liquid material expelled from achachairu plantmaterial as a result of pressing. Juice can contain solid particles,semi-solid particles, and/or droplets of water-immiscible liquids of avariety of sizes (collectively referred to as “achacha particles”) in anaqueous serum. Achachairu particles, based on size and ease of removal,can be qualitatively described as either “pulp” or “cloud”. Whiledetails depend on properties of the involved materials (e.g. whole fruitversus selected parts) and exact processing parameters, an example of asize boundary between “pulp” and “cloud” could lie between about 1 andabout 100 microns.

“Pulp” refers to relatively large achachairu particles present in juiceand those same achachairu particles removed from the juice by mechanicalseparation. It is not uncommon to see and distinguish individual pulpparticles with a naked eye. Pulp particles suspended in the juice areamenable to removal by mechanical separation, including, but not limitedto, sedimentation by ambient gravity, skimming, passing through a meshor a filter, or centrifugation.

“Cloud” refers to relatively small achachairu particles present in theachachairu juice, and may even include dissolved compounds (e.g., highmolecular weight compounds such as proteins and polysaccharides), whichcan be readily induced (e.g., by coagulation or temperature change) toform particles. Cloud particles are typically visible as turbidity ofthe achachairu juice, and individual cloud particles are generallyindistinguishable to the naked eye. Cloud particles are generallydispersed in the achachairu juice and such dispersions tends to remainstable for a longer period time than a pulp suspension, especiallycolloidal components of the cloud. Removal of cloud particles bymechanical means such as those used for pulp removal can be difficult.

“Destabilizing treatment” generally involves exposing a material toelectromagnetic waves in order to modify one or more physical propertiesof the material (e.g., the real component of low-frequency dielectricconstant). With regard to achachairu juice, destabilization treatmentdegrades stability of the particle dispersion in the juice by causingagglomeration and/or aggregation of particles (especially cloudparticles) into assemblies that are sufficiently large and stable toenable and/or improve their subsequent removal by mechanical separationtechniques such as those described above for pulp removal.

“Serum” refers to liquid portion of achachairu juice remaining afterpulp and cloud, as well as possible contaminants of concern, has beenremoved. Achachairu serum is free or substantially free of achachairuparticles.

“Finished ingredient” refers to serum, extract, and/or particle-freeextract that is in a form suitable for subsequent sale and/orincorporation into a personal care product. The finished ingredient mayinclude preservatives that protect the serum, extract, particle-freeextract, and/or compositions containing the same against environmentalchallenges such as temperature, atmosphere (e.g., oxygen), light, andmicroorganisms. It is to be appreciated that an ingredient free ofpreservatives is also contemplated herein. Surprisingly, it has beenfound that the processes described herein produce finished ingredientswith multifunctional biological activities that are either free of orsubstantially free of benzophenones and proteins.

“Post-destabilization precipitate” refers to achachairu particlesremoved from the juice via mechanical separation following destabilizingtreatment.

“Solvent extraction” in FIG. 2 involves conducting an extraction onachachairu particles removed from the achachairu juice with one or moresolvents (e.g., dipropylene glycol).

The extract shown in FIG. 2 refers to the solution resulting from thesolvent extraction of the achachairu pulp and post-destablizationprecipitate. In some instances, any achachairu particles remaining inthe extract may be separated (e.g., mechanically) to yield aparticle-free extract. The extract and/or particle-free extract mayserve as a finished ingredient, or as a base for a finished ingredientvia addition of preservatives.

“Post-extraction precipitate” refers to achachairu particles separatedfrom the extract in FIG. 2.

Achachairu Serum Fraction

The compositions herein may include an achachairu serum fraction and/ora salt, isomer, or derivative thereof. Serum fraction techniques involveseparating the fresh cell juice found in plant material from the rest ofthe plant matter and processing the juice to provide a stable, refinedserum. Examples of achachairu serum fractions that may be suitable foruse in the compositions and methods herein include Recentia® GH (wholefruit) and Recentia® GH-P (peel only) from Ashland SpecialtyIngredients. Achachairu peel serum fraction has the INCI designation“Garcinia humilis Peel Extract” and the CAS No. 1622986-60-0.

The achachairu serum fractions herein may be prepared from freshachachairu using a method that helps maintain the integrity of thebioactive components present in the achachairu plant material. Careshould be taken to preserve the achachairu plant material integrityduring harvesting and transport, so as to minimize environmental factorssuch as moisture loss and biological degradation. All steps of the serumfraction making process should be completed in the shortest possibleperiod of time to minimize exposure of the fresh achachairu plantmaterial to sun, high temperature, and other undesirable environmentalfactors. Harvesting (e.g., by hand or mechanical cutting) should beconducted in a manner that avoids or minimizes the chopping, mashing,crushing, or other type of injury to the achachairu plant material to beused (e.g., fruit). Harvest and transport of achachairu plant materialshould be conducted in a manner to avoid moisture loss or spoilage.After harvesting, the achachairu plant material (e.g., fruit, leaves) iscleaned to remove debris prior to further processing. Cleaning isperformed under conditions to prevent the initiation of the release ofthe juice from the fruit, to cause injury, or to remove valuablecomponents. For example, the harvested achachairu plant material may bewashed with water at low-pressure (e.g., 1 kg/cm² or less) for a shortduration (e.g., 5 minutes or less). Excess water is removed from washedfruit biomass before processing.

The washed plant material is processed (e.g., macerated and pressed) toyield juice. In some instances, the whole fruit or a portion thereof(e.g., peel, fruit flesh, and/or seed) is macerated (e.g., by grinding)and then pressed to separate the juice from the rest of the plantmatter. For example, a hammer mill may be used to grind the whole fruitto yield plant material particles of a desired size (e.g., less than orequal to 0.5 cm) in a short time (e.g., 10 seconds or less) and withoutsignificant increase of biomass temperature (e.g., 5° C. or less). Theseparation of juice from the achachairu plant material is commencedpromptly, as soon as possible, after maceration of the achachairu plantto avoid a significant increase in temperature of the juice and/or pulp.For example, macerated achachairu leaves can be pressed using ahorizontal, continuous screw press, wherein the pressure on the cone ismaintained at 24 kg/cm², screw speed is at 12 rpm, and biomasstemperature increase is 5° C. or less.

The achachairu juice can be subjected to further processing immediately,or the juice may be frozen at about −30° C. and thawed and processedlater.

Achachairu juice typically contains a variety of achachairu particles(i.e., pulp and cloud) in an aqueous serum. Thus, in order to obtain thedesired serum fraction, all or substantially all of the achachairuparticles should be removed from the juice. In some instances, it may bedesirable to provide a serum fraction that includes less than 25% drymatter (e.g., less than 20%, 15%, 12%, 10% or even less than 9% drymatter). The pulp may be removed from the achachairu juice using asuitable mechanical separation technique such as, for example,straining, filtration (including filtration utilizing a pressuregradient), skimming, sedimentation by ambient gravity, decanting,centrifugation, and/or a combination of these to yield pulp-free orsubstantially pulp-free juice. The cloud may be removed by exposing thejuice to electromagnetic waves to destabilize the cloud dispersion inthe juice. The frequency of electromagnetic waves may range between 300MHz and 50 GHz. Suitable devices for generating such electromagneticwaves include, but are not limited to magnetrons, power grid tubes,klystrons, klystrodes, crossed-field amplifiers, travelling wave tubes,and gyrotrons. In some instances, a magnetron operating at a frequencyof 915 MHz, 2.45 GHz, and 5.8 GHz can allow the value of real componentof low-frequency dielectric constant (ε′₀) to be decreased during thetreatment by between 10 and 40 compared to its value prior to treatment.The value of real component of low-frequency dielectric constant (ε′₀)can be determined using broadband dielectric spectroscopy data obtainedvia equipment and software from Agilent Technologies: PNA-L NetworkAnalyzer N5230C with 85070E dielectric probe kit, N4693-60001 electroniccalibration module, and 85070 software. The calculation is performedaccording to method described in the article Cole, K. S., & Cole, R. H.(1941). Dispersion and absorption in dielectrics I. Alternating currentcharacteristics. The Journal of Chemical Physics, 9(4), 341-351.

Other non-limiting examples of techniques for providing serum fractionsare disclosed in U.S. Pat. Nos. 7,442,391 and 7,537,791 to Koganov andU.S. Pub. No. 2012/0201768, filed by Swanson, et al.

Achachairu Extract

The compositions and methods herein may employ an achachairu extract. Anachachairu extract is obtained by separating compounds from achachairuplant matter with an exogenous solvent. Consistent with the generalprinciple of “like dissolves like,” the choice of extraction solventlargely determines the type and number of compounds that will resultfrom any particular extraction technique. For instance, polar compoundsare typically extracted out by using a polar solvent, while non-polarcompounds are extracted out by using a non-polar solvent. Thecorrelation between solvent polarity and the types of materials isolatedusing traditional solvent extraction is described in Houghton & Raman,Laboratory Handbook for the Fractionation of Natural Extracts (1998).

The achachairu extracts herein may be obtained using any suitableextraction technique known in the art. In some instances, the achachairuextract may be obtained by the following procedure: (i) place thedesired portion of dried plant material (e.g., whole fruit, fruit pulp,peel, seeds, stem, bark, leaves) in a conical glass percolator; (ii) addthe indicated percentage of extraction solvent in a w/w ratio of 1 partplant material to 2 parts extraction solvent (when the indicatedpercentage of extraction solvent is less than 100%, the remainingsolvent is water (e.g., 95% ethanol with 5% water, 50% ethanol with 50%water)); (iii) allow the extraction to proceed for 16 to 24 hours; (iv)collect the percolate, and repeat the above process until the resultingpercolate is substantially free from plant additional extract; (v)combine the percolates, evaporate to dryness under reduced pressure, andstore the resulting extract under nitrogen at less than 4 degreesCelsius. Extracts may be used without any further modification or may bemodified (e.g., ethoxylated, esterified) to form a derivative material.

Achachairu Powder or Juice

The personal care compositions for use with the methods herein mayinclude an achachairu powder or juice. Achachairu juice may be obtainedby macerating and process achachairu plant material, for example, asdescribed hereinabove. Achachairu powder may be obtained by dryingachachairu plant material and processing the dried material intoparticles. Additionally or alternatively, the achachairu plant materialmay be processed into particles and then dried. Processing theachachairu into particles may include one or more grinding stepsperformed before and/or after drying. The achachairu biomass may bedried and processed into particles using any suitable method known inthe art. The achachairu powder may have a weight average particle sizeof from 50 to 750 microns. Once the achachairu is processed intoparticles of the desired size, the powder can be incorporated into apersonal care composition using conventional methods for making suchcompositions.

Dermatologically Acceptable Carrier

The skin care compositions herein include a dermatologically acceptablecarrier at an amount of 20% to 99.99% (e.g., 50% to 99%, 60% to 98%, 70%to 95%, or even 60% to 80%) by weight of the composition. The carriermay be aqueous or anhydrous. The form of the carrier is not particularlylimited, and can be any suitable form known in the art for theapplication desired (e.g., solutions, dispersions, emulsions andcombinations thereof). “Emulsions” refer to compositions having anaqueous phase and an oil phase. Emulsion carriers include, but are notlimited to oil-in-water, water-in-oil and water-in-oil-in-wateremulsions. Emulsion carriers herein may include from 0.01% to 10% (e.g.,0.1% to 5%) of an emulsifier (e.g., nonionic, anionic, cationicemulsifier, or a combination thereof). Suitable emulsifiers aredisclosed in, for example, U.S. Pat. No. 3,755,560, U.S. Pat. No.4,421,769, and McCutcheon's Detergents and Emulsifiers, North AmericanEdition, pages 317-324 (1986).

Optional Ingredients

The compositions of the present invention may contain a variety ofoptional ingredients that are conventionally used in skin carecompostions, as long as the optional ingredient(s) do not undesirablyalter product stability, aesthetics or performance The optionalingredients, when incorporated into the composition, should be suitablefor contact with human skin without undue toxicity, incompatibility,instability, allergic response, and the like within the scope of soundjudgment. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992)describes a wide variety of nonlimiting cosmetic and pharmaceuticalingredients. The compositions herein may include 0.0001% to 50%; from0.001% to 20%; or even 0.01% to 10%, by weight of the composition, ofoptional ingredients. Some non-limiting examples of optional ingredientsinclude vitamins, minerals, peptides and peptide derivatives, sugaramines, oil control agents, flavonoid compounds, anti-oxidants and/oranti-oxidant precursors, preservatives, phytosterols, proteaseinhibitors, tyrosinase inhibitors, anti-inflammatory agents,moisturizing agents, emollients, humectants, exfoliating agents, skinlightening agents, sunscreens, sunless tanning agents, pigments, filmformers, thickeners, pH adjusters, opacifying agents,colorings/colorants, particles, fragrances, essential oils, lubricants,anti-acne actives, anti-cellulite actives, chelating agents,anti-wrinkle actives, anti-atrophy actives, phytosterols and/or planthormones, N-acyl amino acid compounds, antimicrobials, antifungals, andcombinations of these. Other non-limiting examples of skin care activescan be found in U.S. Pub. Nos. 2010/0272667 and 2008/0206373 and U.S.Pat. No. 8,790,720.

Methods of Regulating a Skin Condition

Skin care compositions comprising an effective amount of achachairu canbe used to regulate a skin condition by topically applying the skin carecomposition to a portion of mammalian tissue in need of treatment orwhere treatment is desired. Identifying a target portion of skin may bebased on, for example, the presence of a visible condition (e.g.,dryness, undesirable feel, redness, inflammation, discoloration, orwrinkles). In some instances, the target portion of skin may not exhibitvisible signs of a skin condition, but a user may still wish to targetsuch an area if it is one that is known to develop a condition (e.g.,skin surfaces that are typically not covered by clothing).

Skin care compositions containing an effective amount of achachairu maybe applied once a day, twice a day, or on a more frequent daily basis,during a treatment period. The treatment period is ideally of sufficienttime for the achachairu to provide the desired benefit. For example, thetreatment period may be of sufficient time for the achachairu to providea noticeable and/or measurable improvement in a hair or skin condition.The treatment period may last for at least 1 week (e.g., about 2 weeks,4 weeks, 8 weeks, or even 12 weeks). In some instances, the treatmentperiod will extend over multiple months (i.e., 3-12 months) or multipleyears. In some instances, a cosmetic composition containing an effectiveamount of achachairu may be applied most days of the week (e.g., atleast 4, 5 or 6 days a week), at least once a day or even twice a dayduring a treatment period of at least 2 weeks, 4 weeks, 8 weeks, or 12weeks.

The skin care compositions herein may be applied locally or generally.In reference to application of the composition, the terms “localized”,“local”, or “locally” mean that the composition is delivered to thetargeted area while minimizing delivery to skin surfaces where treatmentis not needed or desired. While certain embodiments herein contemplateapplying a composition locally to an area, it will be appreciated thatthe compositions herein can be applied more generally or broadly to oneor more skin surfaces. In certain embodiments, the compositions hereinmay be used as part of a multi-step beauty regimen, wherein the presentcomposition may be applied before and/or after one or more othercompositions.

Using the skin care compositions herein according to the present methodscan provide a skin benefit. Some non-limiting examples of skin benefitsinclude reducing the appearance of wrinkles, deep lines, fine lines,crevices, bumps, large pores; increasing the convolution of thedermal-epidermal border; skin lightening; increasing elasticity,decreasing sagging, reducing cellulite; reducing the appearance ofunder-eye circles, reducing the appearance of discoloration, reducinghyperpigmentation, increasing skin luminosity, and combinations thereof.

The skin care compositions may be applied by any suitable means knownfor applying such products, including rubbing, wiping or dabbing withhands, fingers and/or an implement. Non-limiting examples of implementsinclude a sponge or sponge-tipped applicator, a swab (for example, acotton-tipped swab), a pen optionally comprising a foam or spongeapplicator, a brush, a wipe, and combinations thereof. The compositionmay be pre-applied to the applicator and, for example, delivered to theuser pre-packaged as such, or the user may be instructed to apply thecomposition to the applicator prior to use. In some instances, thecomposition may be stored in an implement, for example, in a separatestorage area for the composition. In this example, the composition maybe transferred to the applicator from the storage area, for example, bysqueezing and/or breaking or by other suitable means. The compositionmay be applied to the keratinous tissue by contacting the applicator andcomposition to the skin. Contact may include, for example, lightpressure, dabbing, rubbing, wiping, or any other suitable means. Whentargeted application is desired, the composition may be applied to thedesired area of keratinous tissue.

Methods for Determining the Effectiveness of Achachairu

Irritation and inflammation in human skin can lead to visible signs ofskin aging (e.g. discoloration and wrinkles), decreased mechanicalstrength, decreased protective functions, and lessened ability torecover from stress and injuries. Ubiquitous stressors such as sunlightand surfactants can be especially problematic. Mitigating irritation andinflammation of the skin, particularly those caused by common stressors,is important and desirable.

The adverse effects of light, most commonly sunlight (though artificialsources are included), on human skin are well known. Overly highexposure to sunlight may cause acute adverse reaction involvingirritation and inflammation, such as sunburn. Exposures insufficient tocause acute reactions can still trigger inflammation-related processes.Accumulated inflammatory damage from sunlight exposure causesdegradation of skin resilience and development of an undesirableappearance (i.e., photoaging).

Surfactants are also known to cause adverse reactions on human skin.Surfactants are used in a variety of personal care and cleansingproducts to allow or improve processes of cleansing, foaming,emulsifying, solubilizing, and dispersing. Repetitive contact withsurfactant-containing products has been shown to cause damage of theskin barrier due to surface or interface activities of the surfactants,which can be perceived by consumers as dryness, itchiness, swelling,redness, and/or pain. The weakened barrier subsequently leads to deeperpenetration of the surfactants into skin and induced irritation andinflammation. As surfactants are widely used in hand soaps, facial andbody washes, shampoos and conditioners, as well as dish, laundry andhousecleaning detergents, human skin contact with surfactants is common.

Irritation and inflammation are commonly viewed as a “cascade”proceeding from necessary release of a signaling compound Interleukin(IL) 1-alpha (or IL-1α) to induction of other downstream cytokines andchemokines such as interleukins IL-6 and IL-8 or other signalingmolecules (Weiss T, Basketter D A, Schröder K R. In vitro skinirritation: facts and future. State of the art review of mechanisms andmodels. Toxicol In Vitro 2004; 18 (3): 231-43). However, previouslypublished data suggest that “cascade” view might not be a comprehensivemodel.

It has been shown that sodium dodecyl sulfate (SDS), a single compoundcommonly used as a benchmark source of surfactant stress in both invitro and in vivo studies, can trigger different portions of theirritation and inflammation process without significantly affectingrelease of a primary cytokine such as IL-1α, depending on concentration.The complexity of irritation and inflammation response of skin cellsimplies that signaling “network” model is a more adequate analogy than asignaling “cascade” model. This indicates that mitigation of such acomplex signaling process must affect more than one pathway, such as byusing a multifunctional bioactive ingredient.

One of the methodologies for studying and quantifying irritation andinflammation includes culturing cells of the tissue most likely to comein contact with stress sources, such as viable epidermal keratinocytesfrom human skin. Human epidermal keratinocytes (HEK) have become thefocus of attention in irritant-induced skin inflammation by virtue oftheir epidermal location, importance in maintaining the integrity of thestratum corneum barrier, and the ability to produce a variety ofinflammatory mediators. Keratinocytes can release a variety ofsignalling substances (e.g., interleukins and arachidonic acidmetabolites) in response to a range of irritants, including surfactantsand sunlight. The amounts of these signalling substances present in atissue sample can be measured via techniques such as Enzyme-LinkedImmunosorbent Assay (ELISA). Bioactive substances that reduce therelease of inflammatory signalling substances may help control the signsof irritation and inflammation in human skin.

Besides signalling substances, other very important compounds inprocesses of irritation and inflammation are those that directly causethe damage. Especially notable inflammatory damage substances are freeradicals (especially reactive oxygen species) and protein-degradingenzymes (proteases). It is possible to detect the presence or measurethe activity of such damaging substances by incubating them with asubstrate they can alter or degrade. The alteration or degradation ofthe substrate can be measured directly (e.g., by loss or development ofcolor or fluorescence) or indirectly (e.g., by ELISA). In some cases,the presence or activity of damaging substances can be measured directlyor indirectly, for example, with spectroscopic techniques (e.g.,Electron Paramagnetic Resonance techniques and the use of chromogenicartificial stable free radicals like DPPH).

The tests selected for determining the effects of serum fractions andextracts of achachairu, and skin care compositions containing the same,include inhibition of proteases (damaging compounds) in non-cell-basedbioassays, inhibition of chemokines, cytokines and prostaglandins(signalling compounds) in cultured human skin cells, inhibition of acompound capable of both signalling and causing damage in cultured humancells, and measuring release or lack thereof of substances indicatingcell damage or sensitization. These tests illustrate safety (e.g., via alack of cytotoxicity and sensitization potential) and the efficacy andpotency of achachairu in inhibiting signalling and damaging compoundsinvolved in skin irritation, inflammation, and aging processes. Thecells in the assays described herein are normal human adult epidermalkeratinocytes (HEK), unless specifically stated otherwise.

EXAMPLES

The following examples are provided for the purpose of illustration onlyand should in no way be construed as being limiting, but rather shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1—Preparation of a Serum Fraction from Achachairu Whole Fruit

In this example, achachairu (Garcinia humilis) whole fruit are used toproduce a serum fraction. The achachairu fruit are harvested fresh fromtrees, inspected, and cleaned prior to processing. Unsound fruit is notused. Selected whole fruit are ground, pressed, and mechanicallyseparated to provide juice and pulp. The yield of juice from achachairuwhole fruit after grinding, pressing and mechanical separation is about60 to 69% weight/weight; the pH of juice from achachairu whole fruit isfrom 3.0 to 3.3. The juice is immediately subjected to destabilizingtreatment by electromagnetic waves in a continuous flow system thatincludes magnetrons operating at a frequency of 5.8 GHz. The parametersof the electromagnetic waves in the destabilization step are set toachieve the decrease in value of real component of low-frequencydielectric constant (E) during the treatment by about 20 Farads permeter (F/m) compared to its value prior to treatment. The de-stabilizedjuice is immediately pumped through a continuous flow centrifuge toyield a serum and post-destabilization precipitate. The serum issubstantially free of benzophenones and protein.

Example 2—Preparation of Serum Fraction from Achachairu Peel

In this example, achachairu fruit peel is used to produce a serumfraction. The achachairu fruit are harvested fresh from trees,inspected, and cleaned prior to processing. Unsound fruit is not used.The fruit peel is separated from whole fruits and then ground, pressed,and mechanically separated to produce juice and pulp. The yield of juicefrom achachairu fruit peel after grinding (maceration), pressing andmechanical separation is about 25 to 35% weight/weight; the pH of juicefrom achachairu fruit peel is 2.7 to 3.0. The juice from fruit peel isimmediately subjected to destabilizing treatment by electromagneticwaves in a continuous flow system that includes magnetrons operating ata frequency of 2.45 GHz. The parameters of the electromagnetic waves inthe destabilization step are set to achieve the decrease in value ofreal component of low-frequency dielectric constant (ε′₀) during thetreatment by about 30 Farads per meter (F/m) compared to its value priorto treatment. The de-stabilized juice is immediately pumped through acontinuous flow centrifuge to yield a serum and post-destabilizationprecipitate. The serum is free or substantially free of benzophenonesand protein.

Example 3—Preparation of Serum Fraction from Achachairu Fruit Flesh andSeeds

In this example, achachairu fruit flesh and seeds are used to produce aserum fraction. The achachairu fruit are harvested fresh from trees,inspected, and cleaned prior to processing. Unsound fruit is not used.The fruit flesh and seed are separated from whole fruits and thenground, pressed and mechanically separated to produce juice and pulp.The yield of juice from Achachairu (Garcinia humilis) fruit flesh andseeds after grinding, pressing and mechanical separation is about 30 to40% weight/weight; the pH of juice from achacha fruit flesh and seeds is3.8 to 4.2. The juice is immediately subjected to destabilizingtreatment by electromagnetic waves in a continuous flow system thatincludes magnetrons operating at a frequency of 2.45 GHz. The parametersof the electromagnetic waves in the destabilization step are set toachieve the decrease in value of real component of low-frequencydielectric constant (ε′₀) during the processing by about 25 Farads permeter (F/m) compared to its value prior to treatment. The de-stabilizedjuice is immediately pumped through a continuous flow centrifuge toyield a serum and post-destabilization precipitate. The serum is free orsubstantially free from benzophenones and protein.

Example 4—Preparation of Extracts from Achachairu Fruit by SolventExtraction

Solvent extractions were conducted with pulp particles, withpost-destabilization precipitate particles, including cloud, or withtheir combination using one or more solvents under particular extractionconditions: type of solvent(s), ratio of solvent to plant material(pulp, or post-destabilization precipitate, or their combinations),extraction temperatures, agitation, time of exposure, etc., in order toextract materials that are soluble in the particular solvents. The ratioof pulp and/or post-destabilization precipitate to solvent can rangefrom 1:2 to 1:1000. Extraction temperature can range from 4° C. to 100°C. Time of the exposure may vary from about 15 minutes to about 96 hrs.

Example 5—Skin Care Composition Formulations

Table 1 provides examples of topical skin care compositions comprisingachachairu. The compositions in Table 1 are made by blending the A phasecomponents with a suitable mixer (e.g., Tekmar RW20DZM) and heating to atemperature of 70-80° C. and maintaining the temperature while stirring.Separately, blend the B phase components with a suitable mixer and heatto 70-75° C., maintaining temperature while mixing. Phase B is added toPhase A while mixing well to emulsify. The emulsion is then milled usinga suitable mill (e.g., Tekmar T-25) for 5 minutes. When the emulsion isat 60° C., phase C is added while continuing to mix. At 40° C., theingredients of phase D and E are added to the emulsion. The emulsion isthen milled using a suitable mill (Tekmar T-25) for 5 minutes resultingin a uniform product.

TABLE 1 Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 Ex. 7 Component % Phase Awater qs qs qs qs qs qs qs glycerol 5.00 7.00 3.00 10.00 5.00 5.00 5.00disodium EDTA 0.10 0.05 0.10 0.10 0.10 0.10 0.10 Phase B IsopropylIsostearate 5.00 2.50 1.33 2.50 5.00 5.00 5.00 Isohexadecane 1.00 1.503.00 1.00 1.00 1.00 1.00 Distearyldimonium 0.00 0.50 1.00 1.50 0.00 0.000.00 Chloride Steareth-2 0.50 2.00 1.00 1.00 0.50 0.50 0.50 cetylalcohol 0.25 0.50 0.32 0.50 0.25 0.25 0.25 tocopherol acetate 0.00 0.500.50 0.50 0.00 0.00 0.00 Steareth-21 0.50 1.00 0.40 0.80 0.50 0.50 0.50stearyl alcohol 0.70 1.50 2.00 2.25 0.70 0.70 0.70 behenyl alcohol 0.801.00 0.40 0.60 0.80 0.80 0.80 ethyl paraben 0.20 0.20 0.20 0.20 0.200.20 0.20 propyl paraben 0.10 0.10 0.10 0.10 0.10 0.10 0.10 polymethyl-1.25 2.50 2.00 0.50 1.25 1.25 1.25 silsesquioxane Phase C Polyethylene1.50 1.00 1.50 2.00 1.50 1.50 1.50 Phase D Water 5.00 10.00 10.00 5.005.00 5.00 5.00 Achachariu serum 0.1 0.5 2.0 10.0 — — — fractionAchachariu extract — — — — 0.1 0.5 2 dexpanthenol 0.25 0.50 0.50 2.000.25 0.25 0.25 Phase E benzyl alcohol 0.25 0.25 0.25 0.25 0.25 0.25 0.25dimethicone/ 0.5 1.00 2.00 0.25 0.5 0.5 0.5 dimethiconol

Example 6—Preservatives

Various preservatives were tested for use with the bioactive serumfractions and bioactive extracts of achacha herein. Table 2 shows anexample of combination/concentrations of preservatives that may beparticularly for preparing finished achacha ingredients (e.g., preparedfrom the serum fractions described in Examples 1, 2, and 3). It has beenfound that the preservatives listed in Table 2 help prevent theformation of precipitates in a finished product.

TABLE 2 Amount Preservative (wt %) Pentylene Glycol (CAS 5343-92-0)1.90% Tetrasodium EDTA (CAS 64-02-8) 0.25% Sodium metabisulfite (CAS7681-57-4) 0.20% Potassium sorbate (CAS 590-00-1) 0.10% Sodium Benzoate(CAS 532-32-1) 0.10% Bioactive Serum Fraction or Extract 97.45%

Example 7—Physico-Chemical Characteristics

Table 3 illustrates methods for testing and evaluating certainphysico-chemical characteristics of achacha serum fractions andextracts. It is to be appreciated that the methods are not limited tothe specific instruments or techniques shown, and equivalent instrumentsand techniques may be used to achieve substantially the same results, asknown by those skilled in the art.

TABLE 3 Property Test Method Units Appearance Determinedorganoleptically. N/A Odor Determined organoleptically. N/A ColorDetermined on Lovibond Comparator 3000 Gardner Scale. Gardner Turn oncomparator lamp. Measure 8 mL of sample into sample scale tube. Inserttube into comparator. Rotate the knobs until two color standards nearestin color to the sample have been located. Record the value of the samplecolor accordingly. If the color of the sample is substantially similarto both, rather than a single standard, then record it as a valuebetween the values of the two standards. Dry Matter Dry matter isdetermined by comparing the weights of liquid % sample with residual drymatter after water has been evaporated. Procedure is based on standardlaboratory practices commensurate with available equipment. Select asuitable aluminum weighing dish (e.g., VWR 25433-016) and weight thedish. Add approximately 4 mL of liquid sample to the dish with the dishon the scale. Determine the weight of the liquid sample by subtractingthe dish weight from the total weight. Repeat this procedure with twoadditional weighing dishes. Place the dishes in a ThermoScientific“Lindberg Blue M” Gravity oven at 105 degrees Celsius for 24 hours.After 24 hours, remove the dishes and allow them to cool forapproximately 5 minutes at room temperature. Weigh each dish. Determinethe weight of the dry sample. Calclulate dry matter percentage bydividing dry sample weight by liquid sample weight. Dry matterpercentage for the sample is the average of the dry matter percentagesfor the three dishes. Refractive index Determined using a ReichertArias ™ 500 brand refractometer nD according to instruction manualsections 6.0, 4.1 and 4.4-4.5. Temperature regulation is provided byCole-Parmer Polystat temperature controller, model number 12108-10 setat 20 C. Automatic Reading Method is enabled. Place 0.5 mL of deionizedwater on the surface of the lower measuring prism, taking care to avoidbubble formation. Close the cell and turn the shadowline adjustment knobto bring the shadowline within the crosshairs. Wait for temperature atrefractometer measuring cell to stabilize, then push Read button. Repeatuntil refractive index of deionized water is determined as 1.333 atleast three times in a row. Rinse the lower and upper surfaces of themeasuring cell with deionized water and blot dry with lint-free wipe.Place 0.5 mL of sample on the surface of the lower measuring prism.Close the cell and turn the shadowline adjustment knob to bring theshadowline within the crosshairs. Wait for temperature at refractometermeasuring cell to stabilize, then push Read button. Repeat until stablereadings have been obtained for sample material at least three times ina row. Record this value as the Refractive Index. Density Determinedwith Densito 30PX densitometer from Mettler g/cm³ Toledo. Procedure isbased on Operating Instructions for Densito 30PX, sections 4 and 6. Setthe instrument display to g/cm³. Calibrate the instrument with 4 cm³ ofdeionized water, avoiding bubble intake or formation. If the density ofthe calibration sample deviates by more than 0.05% from the expecteddensity of water at ambient temperature, recalibrate the densitometer asper Operating Instructions. Eject the calibration sample and fill thesample loops with 4 cm³ of sample, avoiding bubble intake or formation.Record the reading. Eject the sample and repeat steps above foradditional readings, until receiving three matching readings in a row.Record the value of density (specific gravity) for the sample. pHDetermined by measuring on a Denver Instrument Model 250 N/ApH/ISE/conductivity meter with pH/ATC electrode number 300729.1.Procedure is based on manufacturer's 301127.1 Rev. D manual, pages iiand 9 through 12. Use pH 4.01 and pH 7.00 buffers to calibrate the pHmeter. Total Plate Determined as per US Pharmacopoeia XXX, NF25, <61>,CFU/ Count Microbiological Limit Tests gm Mold/Yeast Determined as perUS Pharmacopoeia XXX, NF25, <61>, CFU/ Microbiological Limit Tests gm E.coli Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU/Microbiological Limit Tests gm Salmonella sp. Determined as per USPharmacopoeia XXX, NF25, <61>, CFU/ Microbiological Limit Tests gmStaphylococcus Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU/aureus Microbiological Limit Tests gm Pseudomonas Determined as per USPharmacopoeia XXX, NF25, <61>, CFU/ sp. Microbiological Limit Tests gmBenzophenones, Dal Molin MM, Silva S, Alves DR, Quintão NLM, Delle %Monache F et al.(2012) Phytochemical analysis and antinociceptiveproperties of Garcinia achachariu Rusby (Clusiaceae) seeds. Arch PharmRes 35: 623-631. Protein Amino acid analysis conducted on Hitachi L-8900amino acid % analyzer according the manufacturer's instructions.

Some physico-chemical characteristics of the present achacha serumfractions and extracts are illustrated in Tables 4, 5, and 6 below. Theserum fractions and extracts are prepared according to the methodsdescribed in the corresponding example(s) above. The achacha serumfraction included in the finished ingredient Recentia® GH-P (CASRN#1622986-60-0), which is illustrated in Table 6 below, is preparedaccording to the process described in Example 2.

TABLE 4 Serum Fractions Serums obtained from: Fruit Flesh Whole FruitFruit Peel and Seed Lot GH 0785 Lot GH 1082 Lot GH 1083 Appearance ClearOrange Clear Orange Hazy Yellow Liquid Liquid Liquid Odor CharacteristicCharacteristic Characteristic Color (Gardner Scale) 6.5 8.5 6.5 Drymatter (%) 13.05 11.02 16.3 Refractive index (nD) 1.355 1.352 1.359 pH3.06 2.85 4.03 Density, g/cm³ 1.0612 1.0514 1.0714 Protein <0.07% <0.13%<0.15% Benzophenones  <0.1%  <0.1%  <0.1%

TABLE 5 Extracts Extracts (1 part plant material + 3 part solvent ratio,weight/weight) obtained from: Fruit Peel Fruit Peel Whole FruitExtracted at Whole Fruit Extracted at Extracted at 4° C. Extracted at40° C. 4° C. Lot GH (PE) 40° C. Lot GH (PE) Lot. GH 0841 0837 Lot. GH0842 0838 Appearance Orange Orange Orange Liquid Orange Liquid LiquidLiquid Odor Characteristic Characteristic Characteristic CharacteristicColor (Gardner Scale) 11.0 12.5 11.5 13.5 Refractive index (nD) 1.43151.4291 1.4314 1.4291 Protein <0.046% <0.049% <0.041% <0.046%Benzophenones About 0.1%  <0.1% About 0.1% or  <0.1% higher

TABLE 6 Recentia ® GH-P Test Parameter Result Appearance Clear OrangeLiquid Odor Characteristic Solubility in water Soluble in any ratioColor (Gardner scale)  5-12 Dry matter (%)  9.0-12.1 pH 2.9-3.8Refractive index (nD) 1.349-1.355 Total Plate Count (CFU/g) <100Mold/Yeast (CFU/g) <100 E. coli (CFU/g) Negative/10 g Salmonella sp.(CFU/g) Negative/10 g Staphylococcus aureus (CFU/g) Negative/10 gPseudomonas sp. (CFU/g) Negative/10 g

Example 8—Absorbance Spectra

Absorbance spectra (in wavelength ranges 200-400 nm and 400-1000 nm)were obtained from 100 microliter samples of serum fractions andextracts of achachairu using a Synergy 2 multi-mode microplate reader(BioTek Instruments, Inc) with 96-well black quartz microplate (HellmaAnalytics GmbH). All dilutions were done as volume/volume. Spectra of100 microliter aliquots of respective solvents (ultrapure deionizedwater and fragrance grade dipropylene glycol) were subtracted fromsample spectra. A summary of the absorbance spectra results areillustrated in FIGS. 3A, 3B, 4A, and 4B. FIGS. 3A and 4A illustrateabsorbance spectra of a wavelength range of 200-400 nm. The samples ofachacha serum fractions and extracts analyzed in FIGS. 3A and 3Bincluded GH 0785, GH 1082, and GH 1083. The samples of achacha serumfractions and extracts analyzed in FIGS. 4A and 4B included GH 0837, GH0838, GH 0841, and GH 0842.

Example 9—Biological Activity

Serums and finished ingredients prepared according to variousembodiments herein were evaluated for biological activity of interest.The evaluations included in this example are: cytotoxicity; skinsensitization potential; inhibition of trypsin activity; inhibition ofelastase activity; inhibition of Kallikrein 5; inhibition of IL-6, IL-8,and/or PGE2; ARE activation; melanin synthesis inhibition; andlipogenesis inhibition. The test methods and results of theseevaluations are discussed in more detail below.

Evaluation of Cytotoxicity

Lactate Dehydrogenase (LDH) is a key cytoplasmic enzyme. Presence of LDHoutside the cells at levels above normal background leakage is anindicator of cell damage or cell death. Assays quantifying LDH in cellculture medium are commonly employed to assess potential cytotoxicity.Observation of cells under a microscope that identify cell rupture orchanges in cell morphology can also contribute to the assessment ofcytotoxicity.

Normal human adult epidermal keratinocytes (HEK) and all cell culturesupplies in the cytotoxicity evaluation were obtained from LifeTechnologies Co. (Carlsbad, Calif., USA). The cells were grown and thenmaintained in keratinocyte basal medium 154 (M154) with added humankeratinocyte growth supplements (HKGS) at 37° C. in an atmosphere of 5%CO₂ and used between passages 2 to 4. For the experiments, HEK cellswere trypsinized, seeded in 96-well plates, and grown to ˜80%confluence. HEK were then exposed, or not, to a stress factor, andincubated for 16 hours with or without test articles at variousconcentrations. After incubation, HEK cell supernatant medium sampleswere collected, and levels of LDH were evaluated using Cytoscan™ LDHAssay kit (Catalog #786-210, produced by G-Biosciences, St. Louis, Mo.,USA). Untreated, unstressed HEK cells were lysed using kit-suppliedlysis buffer as a positive control, with lysate used as assay positivecontrol and a measure of maximum LDH release. Lower induced LDH release,when confirmed by microscopy, indicates lower cytotoxicity.

Evaluation of Skin Sensitization Potential

It is possible for a material to not be cytotoxic, and yet be unsafe dueto provoking an allergic reaction upon skin contact. Typically, initialexposure to an allergen sensitizes the immune system, and followingexposures cause an allergic response. Recent progress in understandingthe mechanisms of skin sensitization identified interleukin-18 (IL-18)production in normal human epidermal keratinocytes (HEK) as a usefulbiomarker for skin contact sensitization (Corsini, et al., “Use of IL-18Production In a Human Keratinocyte Cell Line to Discriminate ContactSensitizers from Irritants and Low Molecular Weight RespiratoryAllergens.” Toxicol In Vitro. 2009 August; 23(5):789-96; Teunis, et al.,“Transfer of a Two-tiered Keratinocyte Assay: IL-18 Production byNCTC2544 to Determine the Skin Sensitizing Capacity and EpidermalEquivalent Assay to Determine Sensitizer Potency.” Toxicol In Vitro.2013 April; 27(3):1135-50). IL-18 is considered a suitable in vitroalternative to animal skin sensitization testing methods such as theLocal Lymph Node Assay. Thus, IL-18 production in HEK was evaluated todetermine sensitization potential.

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. After incubation withtest articles or controls for 16 hours, the HEK cells were lysed with100 μl/well of 0.5% Triton X-100 in pH 7.4 Phosphate Buffered Saline(PBS). The cell lysates were collected, and IL-18 was quantified usingHuman IL-18 ELISA Kit (Catalog #7620, produced by MBL International Co.,Woburn, Mass., USA). A known skin sensitizer, para-phenylenediamine(pPD) (positive control), significantly induced IL-18 compared tovehicle control in HEK cultures. Fold changes of IL-18 levels betweentest articles and respective vehicle controls were calculated andcompared to pPD. Lower induction of IL-18 indicates lower sensitizationpotential.

Inhibition of Trypsin Activity

Collagen fibers provide mechanical strength and support for the skin. Aubiquitous protease, trypsin, is associated with damage andinflammation. Trypsin breaks down collagen, potentially leading todecreased mechanical strength of the skin, as well as wrinkles anddarkening after stress or injury (Burns T, Breathnach S, Cox N,Griffiths C. Rook's Textbook of Dermatology. Eighth Edition.Wiley-Blackwell, 2010. Vol. 1 Sections 8.21 to 8.27. Vol. 2 Section29.7).

Trypsin inhibition was determined via an EnzChek kit utilizing caseinsubstrate with intra-molecularly quenched fluorescent label moieties(Catalog # E6638, produced by Life Technologies). Testing was conductedaccording to manufacturer instructions. Digestion buffer concentrate wasdiluted in deionized water. Substrate and bovine trypsin (Sigma catalognumber T9201) were dissolved and diluted in the digestion buffer. Testarticles were dissolved and diluted in digestion buffer. Calibrationcurve was constructed with amounts of trypsin ranging from 1000nanograms to about 1.4 nanograms in reaction volume. Soybean trypsininhibitor, type I-S (Sigma) was used as a positive control.

Amount of trypsin in wells with test articles and controls was fixed at1000 nanograms. IC₅₀ was calculated as concentration of test article inthe reaction volume (e.g. microtiter plate well) necessary to reduce thetrypsin activity to 50%. Lower IC₅₀ values indicate higher potency and adegree of efficacy.

Inhibition of Elastase Activity

Elastin is a protein essential to elastic fiber network contained inconnective tissues which depend on elasticity for their function, suchas skin. Excessive elastase activity, commonly related to inflammation,degrades elastin and decreases strength and resilience of the skin.During inflammatory processes, elastase can be found in areas beyondthose where it is produced or secreted. Human neutrophil elastaseinhibition by test articles was determined in kinetic colorimetric assaydescribed by Elastin Products Company, Inc. (Elastin Products Company.Assay with N-MeO-Suc-Ala-Ala-Pro-Val-pNA (EPC No. FH237) as substrate.Elastin Products Company, Inc. Research Biochemicals Catalogue. 2004. p.84) and modified for its use with 96-well microtiter plates (Corning3641) from Corning, Inc. (and Synergy 2 microplate reader from BioTekInstruments, Inc. The N-Methoxysuccinyl-Ala-Ala-Pro-Val-pNA substrate(EPC, Catalog No: FH237), and elastase (EPC SE563) were from ElastinProducts Company (Owensville, Mich., USA). Working solution of elastasewas prepared with 0.15 M pH 7.5 Tris-HCl buffer containing 50 mM NaCl.Working solution of substrate was prepared in 0.15 M pH 5.0 acetatebuffer containing 100 mM NaCl, with an aliquot of 2% by volume of finalbuffer of 1-methyl-2-pyrrolidone used for initial dissolution of thesubstrate. Deionized water was used to dissolve buffer components.Reaction volume in each well was 224 μl; concentration of elastase was0.87 units/ml, and substrate, 363 μM.

Enzymatic activity in cleaving the substrate was indicated by adevelopment of yellow color measured as an increase in absorbance at 410nm wavelength. The mean of maximum rate of absorbance increase innegative control wells was considered as 100% of enzyme activity. IC₅₀was calculated as concentration of test article in the well whichreduced the elastase activity to 50%. Lower IC₅₀ values indicate higherpotency and a degree of efficacy.

Inhibition of Kallikrein 5

Kallikrein 5 (KLK5), also known as stratum corneum tryptic enzyme, is atrypsin-like serine protease. Recent in vitro and in vivo evidenceimplicates increased levels of KLK5 in augmented inflammatory responsesuch as rosacea (Two A M, Del Rosso J Q, Kallikrein 5-mediatedinflammation in rosacea: clinically relevant correlations with acute andchronic manifestations in rosacea and how individual treatments mayprovide therapeutic benefit. J Clin Aesthet Dermatol. 2014 January;7(1): 20-5) and in induction of atopic dermatitis-like lesions (Briot A.et al., Kallikrein 5 induces atopic dermatitis-like lesions throughPAR2-mediated thymic stromal lymphopoietin expression in Nethertonsyndrome. J Exp Med. 2009 May 11; 206(5):1135-47). Normal human adultepidermal keratinocytes (HEK) were cultured as described above for thecytoxicity evaluation. After incubation with test articles or controlsfor 16 hours, HEK cell culture supernatants were collected. KLK5 wasquantified using a human KLK5 immunoassay Quantikine ELISA kit (Catalog# DKK500, produced by R&D Systems, Minneapolis, Minn.). The changes ofKLK5 concentrations between test articles and vehicle controls werecalculated and compared. IC₅₀ (concentration of test article necessaryto reduce KLK5 levels to 50% compared to samples from untreated cells)values were calculated by sigmoidal curve fitting with SigmaPlot 10.0(Systat Software). Lower IC₅₀ values indicate higher potency and adegree of efficacy.

Inhibition of IL-6 and/or IL-8 Induced by SDS

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. The cells were thenincubated with test articles and/or controls for 16 hours. Presence ofsodium dodecyl sulfate (SDS) in cell cultivation medium at specificconcentrations was used for induction of chemokines and cytokines. IL-8was induced by 6 μg/mL SDS, IL-6 by 12.5 μg/mL SDS. After incubation,HEK cell supernatants were collected. Quantikine® ELISA kits (R&DSystems Inc, Minneapolis, Minn.) were used to quantify theseinterleukins in the supernatants. IL-8 was quantified by Human CXCL/IL-8Immunoassay kit (Catalog # D8000C), and IL-6 was quantified by HumanIL-6 Immunoassay kit (Catalog # D6050). IC₅₀ (concentration of testarticle necessary to reduce interleukin levels to 50%, with samples fromuntreated cells considered as 0% and samples treated solely withrespective inducing quantity of SDS as 100%) values were calculated bysigmoidal curve fitting with SigmaPlot 10.0 (Systat Software). LowerIC₅₀ values indicate higher potency and a degree of efficacy.

Inhibition of IL-6 and/or IL-8 and/or PGE2 Induced by Full-SpectrumSunlight from Artificial Source

Normal human adult epidermal keratinocytes (HEK) were cultured asdescribed above for the cytoxicity evaluation. The cells were washedonce, and M154 was replaced with PBS. Both the washing and thereplacement were done with PBS to remove light-absorbing components ofM154. The 96-well plate containing HEK was then covered withUV-transparent 1 mm quartz sheet, placed on white underlay atopcontrolled Peltier-cooled surface maintaining room temperature, andirradiated with a dose of 20 J/cm² of artificially produced fullspectrum sunlight at dose rate of about 1100 W/m², as measured viapyranometer through same quartz cover. PBS was then removed and replacedwith M154, and cells were incubated with test articles and/or controlsfor 16 hours. Identical manipulations, with exception of presence ofsunlight, were carried out with HEK serving as unstressed controls.Irradiation equipment was obtained from Solar Light Company, Glenside,Pa. and included Solar Simulator LS1000-6R-002 in Airmass 1.5configuration using plain mirror; XPS1000 precision current source, andPMA2144 Pyranometer. After incubation, HEK cell supernatants werecollected. Quantikine® ELISA kits (R&D Systems Inc, Minneapolis, Minn.)were used to quantify interleukins in the supernatants. IL-8 wasquantified by Human CXCL/IL-8 Immunoassay kit (Catalog # D8000C), IL-6was quantified by Human IL-6 Immunoassay kit (Catalog # D6050); and PGE2was quantified using Parameter™ Prostaglandin E₂ Assay (Catalog #KGE004B). IC₅₀ (concentration of test article necessary to reduceinterleukin or prostaglandin levels to 50%, with samples fromnon-irradiated cells considered as 0% and from irradiated cellsconsidered as 100%) values were calculated by sigmoidal curve fittingwith SigmaPlot 10.0 (Systat Software). Lower IC₅₀ values indicate higherpotency and a degree of efficacy.

Table 7 summarizes the results of evaluating inhibition of IL8 and PGE2activity induced by full-spectrum sunlight from an artificial source andcytotoxicity (LDH/microscopy).

TABLE 7 IL8/full spectrum PGE2/full spectrum Cytotoxicity Material suninduction sun induction (LDH/microscopy) Whole Fruit Some inhibition atInhibits at low Cytotoxic at 0.1% Extract, Lot 0841 low concentrationsconcentrations, IC₅₀ and above (~25% inhibition at 0.01% 0.01%) FruitPeel Extract, Inhibition at low Inhibits at low No cytotoxicity at Lot0837 concentrations concentrations, IC₅₀ 0.1% (estimated IC₅₀ 0.04%0.012%, max inhibition 66% at 0.05%) Fruit Peel Serum No significantIC₅₀ between No cytotoxicity at Fraction, Lot 1082 inhibition0.003-0.03%. Maximum 0.1% inhibition 70% at 0.001% Whole Fruit Serum Nosignificant Maximum inhibition No cytotoxicity at Fraction, Lot 0785inhibition 40% at 0.01% 0.1% Fruit Flesh + Fruit No significantNon-significant No cytotoxicity at Seed Serum inhibition inhibition at0.01-0.02% 0.1% Fraction, Lot 1083

Table 8 summarizes the results of evaluating inhibition of IL8 inducedby SDS; KLK5 inhibition; IL18 inhibition; trypsin inhibition; elastaseinhibition; and cytotoxicity.

TABLE 8 IL8 Cytotoxicity inhibition (LDH/ Trypsin Elastase Material(SDS) KLK5 IL18 microscopy) Inhibition Inhibition Achachariu Not testedIC₅₀ Maximum No IC₅₀ IC₅₀ (Garcinia 0.18% 1.46 fold cytotoxicity 0.37%0.26% humilis) Fruit (benchmark at 0.1% Peel Serum sensitizer Fraction,7.8 fold) Lot 0822 Achachariu About 65% IC₅₀ Maximum No IC₅₀ IC₅₀(Garcinia inhibition 0.2% 1.8 fold cytotoxicity 0.32% 0.28% humilis) at0.1% (benchmark at 0.1% Whole Fruit sensitizer Serum 7.8 fold) Fraction,Lot 0786

In summary, biological activity results suggest that preparations ofachachairu: (i) are not skin sensitizers; (ii) are not cytotoxic atselected concentrations that could be relevant to their concentrationsin finished product formulation; and (iii) potential usefulness formitigating signs of skin aging caused by inflammation and relatedprocess (e.g., those triggered by stresses to the skin, includingenvironmental stress such as full-spectrum sun exposure). Morespecifically, the test results suggested the following: (i) againstPGE2, achachairu whole fruit extract is slightly more effective thanfruit peel extract; (ii) against PGE2, achachairu fruit peel serumfraction is more effective than whole fruit serum fraction, which istentatively better than fruit flesh+fruit seed serum fraction; and (iii)achachairu whole fruit serum fraction is effective for inhibitingSDS-induced IL-8 activity, but not against sun-induced IL8, which issurprising. Also surprising was: (i) the potent inhibition demonstratedby fruit peel extract against sun-induced IL8 and fruit peel serumfraction against sun-induced PGE2; (ii) the lack of any notable activityby fruit flesh+fruit seed serum fraction; (iii) fruit peel serumfraction and whole fruit serum fraction are more cytotoxic than fruitflesh+fruit seed serum fraction; and (iv) whole fruit extract is morecytotoxic than fruit peel extract.

Anti-Oxidant Response Element Activation

The ARE is the so-called “master switch” believed to control theantioxidant defense system of most cells. When the ARE is activated inresponse to oxidative stress, the corresponding genes signal the cell tobegin producing reduction/oxidation regulators and/or reactive oxygenspecies (“ROS”) quenching proteins and enzymes. ROS are highly reactivemolecules formed naturally within cells as a natural byproduct of thenormal metabolism of oxygen and play a role in cell signaling andhomeostasis. However, when a cell is exposed to a stressor such as heator UV radiation, ROS levels can increase, and in some instancesdramatically. As the damage caused by ROS accumulates over time, itcauses more and more oxidative stress at the cellular level thatultimately may lead to tissue damage and/or organ dysfunction. Thus,without being limited by theory, it is believed that if achachairu candemonstrate the ability to activate the ARE, then applying an effectiveamount of achachairu to keratinous tissue may help fight cellular damageassociated with oxidative stress.

ARE activation was quantitated using the ARE-32 reporter cell lineavailable from CXR-Biosciences as described below. ARE-32 is a stableMCF7 cell line containing pGL8x-ARE (8 copies of the rat GST ARE linkedto the luciferase gene) and pCDNA3.1, which contains the neomycinselectable marker. Selection was performed in the presence of G418 andresistant clones were isolated. Clones were screened for induction ofluciferase in response to tBHQ.

The ARE-32 cells are maintained routinely in Dulbecco's Modified EagleMedium (phenol red free) (“DMEM”) containing: 10% fetal bovine serum(“FBS”), 50 units/ml penicillin & 50 μg/ml streptomycin, 0.8 mg/ml G418.Cells are subcultured every 3-4 days. If needed, cells can be frozen inmedium that contains 90% FBS and 10% DMSO.

ARE Method

In a 96 well-plate, 1×10⁴ cells/well are seeded in 100 μl DMEMcontaining 50 units/ml penicillin, 50 μg/ml streptomycin, 0.8 mg/ml G418and 10% FBS. Next, the cells are incubated at 37° C. in a 5% CO₂incubator for 24 hrs, and then the medium is replaced with 100 μl freshmedia. The test samples are treated with achachairu serum fractions atthe concentration listed in Table 11 (1 ul per well), the positivecontrol is 25 uM TBHQ. (10 mM tBHQ of stock solution freshly prepared inDMSO). 100 ul of media is added after treatment for a final assay volumeof 200 uL. The cells are incubated at 37° C. in CO₂ incubator foranother 24 hrs. The test samples are then assayed for luciferaseactivity with Steady-glo™ brand assay system according to themanufacturer's instruction.

The results of the test are summarized in Table 9. At concentrations of0.167% to 1.5%, both the whole fruit and peel serum fractions appear toprovide more ARE activation than than the control. The whole fruit alsodemonstrated a directional increase at 0.056% versus the control.

TABLE 9 ARE Activation Whole fruit¹ Peel² % increase p-value vs. %increase p-value vs. v/v % achachariu vs control control vs controlcontrol 1.5 929 0.000087 1133 0.000046 0.5 489 0.0023 614 0.00086 0.167197 0.0076 209 0.0043 0.056 134 .0.091 113 0.67 0.0185 103 0.96 92 0.790.006173 112 0.83 99 0.99 0.002058 93 0.89 92 0.94 0.000686 120 0.78 930.94 ¹Recentia ® GH from AkzoNobel ²Recentia ® GH-P from AkzoNobelMelanin Synthesis Inhibition—B16 Assay

Overproduction of melanin is generally associated with a variety of skinpigmentation conditions (e.g., age spots, vitiligo, solar lentigines,and melasma). Thus, without being limited by theory, it is believed thatif achachairu can demonstrate the ability to inhibit melanin production,then applying an effective amount of achachairu to skin may help improvethe appearance of skin pigmentation conditions.

A commercially available B16-F1 mouse melanoma cell line from AmericanTissue Culture Collection, Virginia, USA was employed in a conventionalmelanin synthesis inhibition assay. The cell culture medium used in theassay is 500 mL of Dulbecco's Modified Eagle's Medium (“DMEM”), 50 mLFetal Bovine Serum (“FBS”), and 5 mL of penicillin-streptomycin liquid.B16-F1 cells that are cultured in this medium and grown to greater than90% confluency will synthesize melanin. While not intending to be boundby theory, it is hypothesized that melanin synthesis is stimulated bythe culture medium and/or stress induced by growth to a high confluency.The DMEM and FBS can be obtained from American Tissue Culture Collectionand the penicillin-streptomycin liquid can be obtained from Invitrogen,Inc., California, USA. Equipment used in the assay include a CO₂incubator (e.g., a Forma Series Model 3110 by Therma Scientific,Massachusetts, USA or equivalent); a Hemocytometer (e.g., Bright Linemodel by Hauser Scientific, Pennsylvania, USA or equivalent); and aUV-Visible Spectrum Plate Reader (e.g., SpectraMax250 from MolecularDevices, California, USA or equivalent).

Day 0: To begin the assay, the cell culture medium is heated to 37° C.and 29 mL of the medium is placed into a T-150 flask. Approximately1×10⁶ of B16-F1 passage 1 mouse cells are added to the T-150 flask andincubated for 3 days at 37° C., 5% CO₂, 90% relative humidity, until˜80% confluency.

Day 3: The cells from the T-150 flask are trypsinized, and the number ofcells is determined using the Hemocytometer. Initiate a 96 well platewith 2,500 cells per well in 100 μL of cell culture medium. Incubate theplate at 37° C., 5% CO₂, 90% relative humidity for 2 days until at least20% to 40% confluent.

Day 5: Remove the cell culture medium from the plate and replace withfresh culture medium (100 uL per well). Add 1 uL of test compounddiluted in a water solvent. Multiple dilution ratios may be tested inorder to generate a dose response curve, wherein preferably three wellsare treated with each dilution ratio. Positive and negative controls mayinclude wells having the cell culture medium, B16-F1 cells, and thesolvent (negative control), and wells comprising the cell culturemedium, B16-F1 cells and and a known melanin inhibitor (e.g.,deoxyarbutin or kojic acid).

Day 7: Cells should have greater than ˜90% confluency. If not, this datapoint is not used. Add 100 uL of a 0.75% sodium hydroxide solution toeach well. Read the 96-well plate using the UV-Vis Plate Reader at 410nm to optically measure the amount of melanin produced between wellsthat are treated with the fava bean extract and control wells that arenot. Wells in which melanin is produced appear brownish in color. Wellsin which little melanin is produced appear clear to light purple incolor. Percentage of melanin synthesis inhibition is calculated by thefollowing equation:

$\frac{100 - {\left\lbrack {{{OD}\; 410\mspace{14mu}{Test}\mspace{14mu}{Compound}} - {{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 2}}} \right\rbrack \times 100}}{\left( {{{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 1}} - {{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 2}}} \right)}$

Where OD410 is the Optical Density at 410 nm as measured by the UV-VisSpectrum Plate Reader.

When Control #3 is used, the formula for percentage melanin synthesisinhibition is:

$\frac{100 - {\left\lbrack {{{OD}\; 410\mspace{14mu}{Test}\mspace{14mu}{Compound}} - {{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 3}}} \right\rbrack \times 100}}{\left( {{{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 1}} - {{OD}\; 410\mspace{14mu}{Control}\mspace{14mu}{\# 2}}} \right)}$

The concentration of test agent needed to provide the IC 50 is recorded.

The results of the test are summarized in Table 10, which shows thatachachairu inhibits melanin synthesis, and thus is expected to provide askin lightening benefit.

TABLE 10 Melanin Synthesis Inhibition B16 (IC 50) Concentration Neededfor IC 50 Composition (v/v %) Achachariu¹ (whole fruit) 0.19 Achachariu²(peel only) 0.19 ¹Recentia ® GH from AkzoNobel ²Recentia ® GH-P fromAkzoNobelLipogenesis Inhibition

This example demonstrates the ability of achachairu to inhibitlipogenesis in human pre-adipocytes. Lipogeneis involves the synthesisof commonly known lipids such as fatty acids and triglycerides, and isone of primary ways mammals store energy. However, lipogenesis alsoinvolves the synthesis of lipids such as sebum. Sebum is a lipidproduced by sebocytes, which are a type of skin cell found primarily inthe sebaceous glands of mammalian skin. Sebum is produced by the body tolubricate and waterproof the skin and hair of mammals. However,overproduction of sebum can result in oily appearing skin and/or skinthat appears to have poor texture. Thus, without being limited bytheory, it is believed that if achachairu can demonstrate the ability toinhibit lipogenesis, then applying an effective amount of achachairu tokeratinous tissue may help regulate conditions associated with sebumoverproduction.

Method

Human pre-adipocytes were selected for use in this example. Because ofthe known difficulty associated with culturing and testing sebocytes,pre-adipocytes are commonly used as a surrogate for sebocytes todetermine the potential of a test agent to inhibit sebum production.

Human subcutaneous pre-adipocytes purchased from Zen-Bio, Inc (Cat. #SP-F-SL) were cultured in PM-1 media (available from Zen-Bio, Inc asCat# PM-1 (plus 5 ng/ml EGF)) to 80-90% confluency. The cells weretransferred to 96-well clear bottom white plates to provideapproximately 40,625 cells/cm² in the well (˜12,500 cells) and 150 μl ofPM-1 media per well, and then cultured for 24-48 hours in a 5% CO₂incubator at 37° C. The PM-1 media was then replaced withdifferentiation medium (Zen-Bio, Inc. Cat# DM-1), and the cells wereincubated for another 6 days. After incubating in the differentiationmedium, 90 μl of the differentiation medium was carefully replaced with140 μl of human subcutaneous adipocyte medium ((Zen-Bio, Inc. Cat#AM-1). Care was taken not to touch or disturb the cells at the bottom ofthe well. 2 μl of achachairu (Recentia® GH-P for AkzoNoble) or controlcomposition (100 μM Genistein (Cat# G6649) from Sigma) was added to eachwell daily for 9 days (total incubation of 15 days). On Day 15, 5 μL ofAdipoRed reagent (Lonza; Cat. Number: PT-7009) was slowly added directlyto cells in the treatment medium, and the plate was gently mixed aftereach row addition. The plate was incubated for 15 minutes at roomtemperature. Lipogenesis was quantitated using an EnVision® brandFluorescent spectrophotometer Plate Reader according to the AdipoRedprotocol. The plates were scanned from the bottom using the 451 mirrorand (excitation 485 nm; emission 535) filter. Each well was scanned in aZ pattern (7 reads across from left to right, 7 reads diagonally fromright to left and 7 reads across from left to right for a total of 21end points).

Percent inhibition was calculated as:

$\frac{{{Average}\mspace{14mu}{Control}\mspace{14mu}{RFU}} - {{Sample}\mspace{14mu}{RFU}}}{{Average}\mspace{14mu}{Control}\mspace{14mu}{RFU}} \times 100$

The cells were assayed and normalized to the control by using aFluoReporter® Blue Fluorometric brand dsDNA Quantitation Kit.Immediately after the screen the AdipoRed containing cell media wasgently aspirated, cells were rinsed with 100 ul 1×PBS taking care not todislodge them from the bottom and 100 μl distilled water was added/well.The plates were frozen at −80° C. to lyse the cells and assayedaccording to the kit instructions at a later date.

The results of the test are summarized in Table 11, which shows thatachachairu inhibits lipogenesis, and thus is expected to help regulateconditions associated with the overproduction of sebum.

TABLE 11 Lipogenesis Inhibition Compostion IC 50 w/v % Recentia ® GH-P(achachariu peel) 0.4% Recentia ® GH (achachariu whole fruit) 0.5%Inhibiting the Cell's Inflammation Response to a Stressor—NF-kappa-beta(“NF-kB”) assay.

NF-kB (i.e., nuclear factor kappa-light-chain-enhancer of activated Bcells) is a protein complex that belongs to the category of“rapid-acting” primary transcription factors (i.e., transcriptionfactors that are present in cells in an inactive state and do notrequire new protein synthesis in order to become activated), whichallows NF-kB to be a first responder to harmful cellular stimuli such asROS and other stressors. NP-kB is found in almost all animal cell typesand is known to be involved in the cellular inflammation pathway.Cellular inflammation is associated with a variety of skin conditions,and thus inhibiting NF-kB activation vis-á-vis cellular inflammationwith an effective amount of achachairu may help treat these types ofskin conditions.

Method

CellSensor™ NF-κB-bla HEK 293T cells (Invitrogen, Cat. # K1165) wereplated in assay medium (DMEM with high glucose (Gibco, Cat. #11965) plus10% dialyzed FBS). The cells were cultured and seeded at 10,000/well in96-well plates (black-sided Poly-D-Lysine coated plates, BD #356692),and then incubated at 37° C. and 95% RH for the 72 hours prior totesting. Recombinant human TNFα (available from R&D systems) was used tostimulate NF-kB activation in the cells. Fisetin (3, 7, 3′,4′-tetrahydroxyflavone) was used to inhibit stimulation of the cells byTNFα. A ToxBLAzer™ DualScreen brand screening kit (Invitrogen, Cat.#K1138) was used as the fluorescent substrate according to themanufacturer's instructions.

The following controls were run on each plate in the assay:High control (Stimulated)=cells+TNFα+1% DMSOBlank (Unstimulated)=cells+1% DMSOStandard (Positive control inhibitor)=cells+TNFα+Fisetin+1% DMSONegative Control (no cells)=assay medium

$\frac{{{High}\mspace{14mu}{Control}} - {Sample}}{{{High}\mspace{14mu}{Control}} - {Blank}} \times 100$NF-κB % inhibition is calculated as:

The results of this test are summarized in Table 12 below. Asillustrated in Table 12, the achachairu whole fruit serum fractions andachachairu peel serum fractions provided an IC 50 for NF-kB inhibitionof 2%. Thus, applying an effective amount of achachairu to a targetportion of keratinous tissue may help regulate conditions related tocellular inflammation.

TABLE 12 Compostion IC 50 (v/v %) Cytotoxicity Recentia ® GH-P 2% Noneobserved (Achachariu Peel only) Recentia ® GH 2% None observed(Achachariu whole fruit)Inhibiting the Cell's Inflammation Response to a Stressor-ProstaglandinE2 (“PGE2”) assay.

PGE2 is a hormone-like substance that is known to participate inmodulation of inflammation. Cellular inflammation is associated with avariety of skin conditions, and thus inhibiting PGE2 activationvis-à-vis cellular inflammation may help treat these types of skinconditions.

Method

Tert keratinocytes (“tKC”) were plated at 40,000 cells/well into 24-wellplates in 1 ml/well volume. EpiLife Medium (Life Technologies cat #MEPICFPRF500) supplemented with keratinocyte growth supplement (Lifetechnologies cat #S-001-5) was used as the assay media. The cells weregrown to confluence/near confluence, and then subjected to 15 mJ/cm²UVB-stress. The test compositions (achachairu and vehicle control)(diluted 1:1000) were added, and the plates were incubated for 18-24hours. The supernatant was removed from each well, and the cells wererinsed with 2 ml/well medium (without supplements). A Cell Titer-Gloassay (measures ATP activity) was conducted on the cells fornormalization. The supernatant was tested in a PGE2 assay (ProstaglandinE2 Assay kit from Cisbio Bioassays cat#62P2APEB) according to themanufacturer's instructions. The PGE2 results were normalized to ATPactivity.

The results of the PGE2 assay are summarized in Table 13. The achachairuwhole fruit serum fraction and achachairu peel serum fractions bothdemonstrated the ability to inhibit release of PGE2 from keratinocytesexposed to 15 mJ/cm2 UVB radiation, which illustrates anti-inflammatoryactivity of achachairu. Thus, a personal care composition comprisingachachairu may be useful for regulating a skin condition related tocellular inflammation.

TABLE 13 PGE2 Release (% of vehicle Test control) StDev Recentia ® GH-PAchachariu peel 28% 8.20% Recentia ® GH (Achachariu whole fruit) 52%11.40% Vehicle control 98% 13.20%

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm”.

All documents cited in the Detailed Description of the Invention are, inrelevant part, incorporated herein by reference; the citation of anydocument is not to be construed as an admission that it is prior artwith respect to the present invention. To the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

Whereas particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of improving the appearance and/or feelof skin, comprising: a. identifying a target portion of skin in need oftreatment or where treatment is desired; and b. applying a skin carecomposition comprising an effective amount of achachairu serum fractionto the target portion of skin during a treatment period, wherein thetreatment period is sufficient for the achachairu serum fraction toprovide a skin care benefit, and wherein the achachairu serum fractionis obtained from achachairu juice that is processed to have a dry mattercontent of less than 25%, by weight of the serum fraction.
 2. The methodof claim 1, wherein the achachairu serum fraction is obtained from apeel of an achachairu fruit.
 3. The method of claim 1, wherein theachachairu serum fraction is substantially free of at least one ofbenzophenone and protein.
 4. The method of claim 1, wherein theachachairu serum fraction is applied to the target portion of skin atleast once per day.
 5. The method of claim 1, wherein the achachairuserum fraction is present in the skin care composition at an amount offrom about 0.01% to about 15%.
 6. The method of claim 1, wherein theskin care composition further comprises about 20% to about 99.99% of adermatologically acceptable carrier.
 7. The method of claim 1, whereinthe skin care composition further comprises at least one additionalactive selected from the group consisting of vitamins, minerals,peptides, sugar amines, anti-oxidants, preservatives, anti-inflammatoryagents, moisturizing agents, skin lightening agents, particles,anti-wrinkle actives, anti-atrophy actives, N-acyl amino acid compounds,and combinations of these.
 8. The method of claim 1, wherein the skincare composition comprises a preservative selected from pentyleneglycol, tetrasodium EDTA, sodium metabisulfite, potassium sorbate, orsodium benzoate.
 9. The method of claim 1, wherein the skin care benefitis selected from the group consisting of an anti-inflammatory benefit,an anti-aging benefit, an anti-oxidant benefit, and a lipogenesisinhibition benefit.
 10. The method of claim 3, wherein the achachairuserum fraction is substantially free of benzophenone and protein.